A Novel Approach to Measure Variability in the Anterior Cruciate Ligament Deficient Knee During Walking: The Use of the Approximate Entropy in Orthopaedics

Objective. The evaluation of variability of biological rhythmic activities through measures such as Approximate Entropy (ApEn) has provided important information regarding pathology in disciplines such as cardiology and neurology. This research lead to the “loss of complexity hypothesis” where decreased variability is associated with loss of healthy flexibility rendering the system more rigid and unable to adapt to stresses. ApEn as a measure of variability and complexity, correlates well with pathology while, in some cases, it is predictive of subsequent clinical changes. The study of human gait could benefit from the application of ApEn since it is also a rhythmical oscillation. Our aim was to assess the variability of the ACL deficient knee, since ACL rupture is a common musculoskeletal injury and is accompanied by altered gait patterns and future pathology in the joint. We hypothesized that the ACL deficient knee will exhibit more regular and less variable walking patterns than the contralateral intact knee. Methods. Ten subjects with unilateral deficiency walked on a treadmill at their self-selected speed, 20% faster, and 20% slower, while kinematics were collected (50 Hz) from 80 consecutive strides for each condition. The ApEn of the resulted knee joint flexion-extension time series was calculated. Results. Significantly smaller ApEn values were found in the ACL deficient knee when compared with the contralateral intact (F = 5.57, p = 0.022), for all speeds. ApEn values significantly increased (F = 5.79, p = 0.005) with increases in walking speed. Conclusions. The altered properties of the ACL deficient knee, which exhibits more regular and less variable patterns than the contralateral intact knee, may decrease the adaptability of the system rendering it less able to adjust to perturbations. This could explain the increased future pathology found in the deficient knee. ApEn can be an important tool in assessing pathology and therapeutic interventions in orthopaedics.     

Genome analysis of two type 6 echovirus (E6) strains recovered from sewage specimens in Greece in 2006

Echovirus 6 (E6) is one of the main enteroviral serotypes that was isolated from cases of aseptic meningitis and encephalitis during the last years in Greece. Two E6 (LR51A5 and LR61G3) were isolated from the sewage treatment plant unit in Larissa, Greece, in May 2006, 1 year before their characterization from aseptic meningitis cases. The two isolates were initially found to be intra-serotypic recombinants in the genomic region VP1, a finding that initiated a full genome sequence analysis. In the present study, nucleotide, amino acid, and phylogenetic analyses for all genomic regions were conducted. For the detection of recombination events, Simplot and bootscan analyses were carried out. The continuous phylogenetic relationship in 2C–3D genomic region of strains LR51A5 and LR61G3 with E30 isolated in France in 2002–2005 indicated that the two strains were recombinants. SimPlot and Bootscan analyses confirmed that LR51A5 and LR61G3 carry an inter-serotypic recombination in the 2C genomic region. The present study provide evidence that recombination events occurred in the regions VP1 (intraserotypic) and non-capsid (interserotypic) during the evolution of LR51A5 and LR61G3, supporting the statement that the genomes of circulating enteroviruses are a mosaic of genomic regions of viral strains of the same or different serotypes. In conclusion, full genome sequence analysis of circulating enteroviral strains is a prerequisite to understand the complexity of enterovirus evolution.     

Assessing computational predictions of the phenotypic effect of cystathionine‐beta‐synthase variants

Accurate prediction of the impact of genomic variation on phenotype is a major goal of computational biology and an important contributor to personalized medicine. Computational predictions can lead to a better understanding of the mechanisms underlying genetic diseases, including cancer, but their adoption requires thorough and unbiased assessment. Cystathionine‐beta‐synthase (CBS) is an enzyme that catalyzes the first step of the transsulfuration pathway, from homocysteine to cystathionine, and in which variations are associated with human hyperhomocysteinemia and homocystinuria. We have created a computational challenge under the CAGI framework to evaluate how well different methods can predict the phenotypic effect(s) of CBS single amino acid substitutions using a blinded experimental data set. CAGI participants were asked to predict yeast growth based on the identity of the mutations. The performance of the methods was evaluated using several metrics. The CBS challenge highlighted the difficulty of predicting the phenotype of an ex vivo system in a model organism when classification models were trained on human disease data. We also discuss the variations in difficulty of prediction for known benign and deleterious variants, as well as identify methodological and experimental constraints with lessons to be learned for future challenges.     

Assessment of predicted enzymatic activity of α‐N‐acetylglucosaminidase variants of unknown significance for CAGI 2016

The NAGLU challenge of the fourth edition of the Critical Assessment of Genome Interpretation experiment (CAGI4) in 2016, invited participants to predict the impact of variants of unknown significance (VUS) on the enzymatic activity of the lysosomal hydrolase α‐N‐acetylglucosaminidase (NAGLU). Deficiencies in NAGLU activity lead to a rare, monogenic, recessive lysosomal storage disorder, Sanfilippo syndrome type B (MPS type IIIB). This challenge attracted 17 submissions from 10 groups. We observed that top models were able to predict the impact of missense mutations on enzymatic activity with Pearson's correlation coefficients of up to .61. We also observed that top methods were significantly more correlated with each other than they were with observed enzymatic activity values, which we believe speaks to the importance of sequence conservation across the different methods. Improved functional predictions on the VUS will help population‐scale analysis of disease epidemiology and rare variant association analysis.     

Linking function to global and local dynamics in an elevator-type transporter

Transporters cycle through large structural changes to translocate molecules across biological membranes. The temporal relationships between these changes and function, and the molecular properties setting their rates, determine transport efficiency—yet remain mostly unknown. Using single-molecule fluorescence microscopy, we compare the timing of conformational transitions and substrate uptake in the elevator-type transporter Glt_Ph. We show that the elevator-like movements of the substrate-loaded transport domain across membranes and substrate release are kinetically heterogeneous, with rates varying by orders of magnitude between individual molecules. Mutations increasing the frequency of elevator transitions and reducing substrate affinity diminish transport rate heterogeneities and boost transport efficiency. Hydrogen deuterium exchange coupled to mass spectrometry reveals destabilization of secondary structure around the substrate-binding site, suggesting that increased local dynamics leads to faster rates of global conformational changes and confers gain-of-function properties that set transport rates.

Transporters are integral membrane proteins that move solutes across lipid bilayers. They undergo concerted conformational changes, allowing alternate exposure of their substrate-binding sites to external and internal solutions (1). In each of these so-called outward- and inward-facing states (OFS and IFS, respectively), further isomerizations accompany substrate binding and release. Transport efficiency depends on the rates of these rearrangements, but linking function and structural dynamics has presented methodological challenges. Single-molecule Forster resonance energy transfer (smFRET)-based total internal reflection fluorescence (TIRF) microscopy (2–4) has been used to monitor the dynamics of the OFS to IFS transitions (5–8) and single-transporter activity (9) in the elevator-type transporter Glt_Ph and other transporters (10–18). Hydrogen–deuterium exchange followed by mass spectrometry (HDX-MS) has been used to pinpoint local changes in structural dynamics in diverse biological systems (19–21). Here, we combine these approaches to link changes in local protein dynamics to the larger-scale conformational transitions and substrate transport in wild-type (WT) and gain-of-function mutants of Glt_Ph.Glt_Ph is an extensively studied archaeal aspartate transporter that is homologous to human excitatory amino acid transporters (EAATs). Structures of Glt_Ph (7, 22–30), and archaeal and mammalian homologs (31–37), show that the transporters assemble into homotrimers via scaffold domains. Each protomer features a mobile transport domain that binds l-Aspartate (l-Asp) and three Na⁺ ions (22, 23, 28, 31, 38) and symports the solutes by an elevator mechanism, moving ∼15 Å across the membrane from an OFS to an IFS (6, 8, 23, 24, 39). During the elevator transitions, two structurally symmetric helical hairpins (HPs) 1 and 2 form the cores of the domain interfaces in the OFS and IFS, respectively (SI Appendix, Fig. S1A) (23, 24, 40). Despite symmetry, they do not have the same function. HP1 is mostly rigid, while HP2 is a conformationally plastic “master regulator” of the transporter, gating substrate in the OFS and IFS and contributing to setting the elevator transition rates (5, 23, 24, 27, 29, 36, 41–47).In this study, we use three previously characterized mutants of Glt_Ph to pinpoint the rate-limiting steps of the transport cycle and probe the protein dynamic properties that correlate with increased transport rates. A K290A mutation at the base of HP1 disrupts a salt bridge with the scaffold domain in the OFS and dramatically increases the elevator dynamics (5, 6). A triple-mutant Y204L/A345V/V366A displays a more modest increase in elevator dynamics and substantially diminished l-Asp affinity (5). Finally, a Y204L/K290A/A345V/V366A mutant combines these substitutions and their effects (5). We compared our previously obtained smFRET data on the elevator dynamics of the WT transporter and the mutants (5) to single-transporter uptake measurements. For WT Glt_Ph, these dynamics and transport measurements established transporter subpopulations that move (5, 6) and work (9) with rates differing by orders of magnitude, with slow transporters dominating the ensemble. We now show that only mutations that both reduce the population of the slow-moving transporters and weaken substrate affinity, such as Y204L/A345V/V366A, reduce the population of the slow-working transporters and confer overall gain-of-function properties. The slow-working population comprises transporters with rare elevator transitions or slow substrate release. We then used HDX-MS to explore how the Y204L/A345V/V366A mutant differed from the WT protein. We found that the mutations decreased the stability of the secondary structure around the substrate-binding site, suggesting that the increased local dynamics underlie reduced kinetic heterogeneity within the mutant transporter ensemble.     

Changes in structural network are associated with cortical demyelination in early multiple sclerosis

The aim of this study was to investigate the interplay between structural connectivity and cortical demyelination in early multiple sclerosis. About 27 multiple sclerosis patients and 18 age‐matched controls underwent two MRI scanning sessions. The first was done at 7T and involved acquiring quantitative T₁ and T₂* high‐resolution maps to estimate cortical myelination. The second was done on a Connectom scanner and consisted of acquiring high angular resolution diffusion‐weighted images to compute white matter structural connectivity metrics: strength, clustering and local efficiency. To further investigate the interplay between structural connectivity and cortical demyelination, patients were divided into four groups according to disease‐duration: 0–1 year, 1–2 years, 2–3 years, and >3 years. ANOVA and Spearman's correlations were used to highlight relations between metrics. ANOVA detected a significant effect between disease duration and both cortical myelin (p = 2 × 10^?8) and connectivity metrics (p < 10^?4). We observed significant cortical myelin loss in the shorter disease‐duration cohorts (0–1 year, p = .0015), and an increase in connectivity in the longer disease‐duration cohort (2–3 years, strength: p = .01, local efficiency: p = .002, clustering: p = .001). Moreover, significant covariations between myelin estimation and white matter connectivity metrics were observed: Spearman's Rho correlation coefficients of 0.52 (p = .0003), 0.55 (p = .0001), and 0.53 (p = .0001) for strength, local efficiency, and clustering, respectively. An association between cortical myelin loss and changes in white matter connectivity in early multiple sclerosis was detected. These changes in network organization might be the result of compensatory mechanisms in response to the ongoing cortical diffuse damage in the early stages of multiple sclerosis.     

Dopamine reuptake transporter–single‐photon emission computed tomography and transcranial sonography as imaging markers of prediagnostic Parkinson's disease

Objective: The objective of this study was to examine whether prediagnostic features of Parkinson's disease (PD) were associated with changes in dopamine reuptake transporter–single‐photon emission computed tomography and transcranial sonography. Methods: Prediagnostic features of PD (risk estimates, University of Pennsylvania Smell Identification Test, Rapid Eye Movement Sleep Behavior Disorder Screening Questionnaire, and finger‐tapping scores) were assessed in a large cohort of older U.K. residents. A total of 46 participants were included in analyses of prediagnostic features and MDS‐UPDRS scores with the striatal binding ratio on dopamine reuptake transporter–single‐photon emission computed tomography and nigral hyperechogenicity on transcranial sonography. Results: The striatal binding ratio was associated with PD risk estimates (P = .040), University of Pennsylvania Smell Identification Test (P = .002), Rapid Eye Movement Sleep Behavior Disorder Screening Questionnaire scores (P = .024), tapping speed (P = .024), and MDS‐UPDRS motor scores (P = .009). Remotely collected assessments explained 26% of variation in the striatal binding ratio. The inclusion of MDS‐UPDRS motor scores did not explain additional variance. The size of the nigral echogenic area on transcranial sonography was associated with risk estimates (P < .001) and MDS‐UPDRS scores (P = .03) only. Conclusions: The dopamine reuptake transporter–single‐photon emission computed tomography results correlated with motor and nonmotor features of prediagnostic PD, supporting its potential use as a marker in the prodromal phase of PD. Transcranial sonography results also correlated with risk scores and motor signs. © 2018 The Authors. Movement Disorders published by Wiley Periodicals, Inc. on behalf of International Parkinson and Movement Disorder Society.